Ge,. Pharmac. Vol. 23, No. 4, pp. 693-700, 1992 Printed in Great Britain.All rightsreserved

0306-3623/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd

ESTROGEN BINDING SITES IN PERIPHERAL BLOOD MONOCYTES A N D EFFECTS OF DANAZOL ON T H E I R SITES IN VITRO KEISUKEWADA,* TOSHIYAITOH, MIKI NAKAGAWA,RYOU MtSAO, HIDEHIROMORI and TERUHIKOTAMAYA Department of Obstetrics and Gynecology, Gifu University School of Medicine, Tsukasa-machi 40, Gifu, Japan (Received 18 November 1991) Abatraet--l. This study was designed to investigate the presence of estrogen type I (high affinity, low capacity) and type II (low affinity, high capacity) binding sites in human peripheral blood monocytes and the effects of danazol on these sites. 2. These two types of estrogen binding sites existed in human peripheral blood monocytes. 3. Danazol bound to these sites in high concentration (10 -6 M, clinical serum concentration during danazol therapy) and decreased the number of both sites. 4. It is suggested that danazol has an anti-estrogenic action to the monocytes through the competition and suppression of estrogen binding sites as seen in the estrogen target organ.

INTRODUCTION Recently it has been well known that the monokines are increased in the peritoneal effusion of patients with pelvic endometriosis. These monokines are secreted from the peritoneal macrophages (Fakih et al., 1987), suggesting that they contribute to progress of pelvic endometriosis and to infertility related with disturbance of fertilization or embryo development in the Fallopian tube (Hill et al., 1987, 1989). Macrophages derived monocytes were stimulated to release interleukin I(IL-I) by estrogen (Polan et al., 1988). Releases of IL-1 and tumour necrotizing factor (TNF) from these macrophages is inhibited by danazol (Nakagawa et al., 1990). The peripheral blood mononuclear cells have been demonstrated to have estrogen binding sites (Ranelleti et al., 1988). The presence of two types of estrogen binding sites, namely estrogen type I and type II binding sites has been proposed in rat uterus (Clark et al., 1979). These two estrogen binding sites are demonstrated in rabbit uterus as well, while danazol, in high concentration (10 -~ M), can bind to estrogen binding sites in vitro and inhibit the estrogen-induced increment of both estrogen binding sites in vivo, paralleling with the decrease of uterine growth (Wada, 1986). These conditions prompts us to investigate the presence of estrogen type I and type II binding sites in the human peripheral blood monocytes and the effects of danazol on those sites. MATERIALS AND METHODS

Subjects The subjects were 10 healthy women aged 20-23yr (21-I-0.9, mean-t-SEM) who had regular menses (28-30 days of cycle). Heparinized blood (200 ml) was obtained *To whom reprint requests should be addressed. 693

from the radial vein at the mid follicular phase determined by basal body temperature, as the normal controls with their consent. Chemicals 2A,6,7-[JH]estradiol/17// ([JH]E2, 95.4Ci/mmol), were obtained from New England Nuclear Corporation, Boston, Mass. F_q, diethylstilbestrol (DES), dihydrotestosterone (DHT), dextran and activated charcoal were from Sigma Chemical Company, St Louis, Mo. Danazol (17a-pregna2,4-dien-20-yno[2,3-d]isoxazol-17-ol)was purchased from Sterling-Winthrop, Rensselaer, N.Y. All other chemicals and reagents were of experimental grade. Harvest of monocytes (macrophages) and incubation Peripheral blood mononuclear cells obtained from the normal women, were separated by Ficoll-Hypaque gradient centrifugation. The monocytes were isolated by adherence to plastic. The 6 ml of aliquot RPMI- 1640 medium (without serum) containing 1-3 x 10~cells were incubated in 60 mm plastic Petri dishes (Falcon, Becton Dickinson & Company, N.J.) for I hr at 37°C in 5% CO2 atmosphere. After removing the nonadherent cells by three washes with the medium, the adherent cells were collected by a cell scraper. 5 subjects were immediately examined radio-receptor assay. Another 5 subjects were divided into two series before the receptor assay. One was incubated with danazol in the concentration of 10-6M for 24hr at 37°C in 5% CO2 atmosphere. Another one was incubated without danazol. The purity of the monocyte preparation was always over 90% as determined by peroxydase stain. Preparation of cytosol and nuclear fractions for [~H]E2 binding Monocytes were resuspended and washed in TEG buffer [1.5 mM ethylenediamine tetra-acetic acid (EDTA), 10 mM Tris-HC1, 10% glycerol, pH 7.4] three times by centrifuging at 800g for 10rain, 4°C. The pellets were resuspended in 2.0ml TEG buffer (5-10 x 10~cells/ml) and disrupted by sonication in a Heat System Sonicator (SONIFIER; Branson, Conn.) at an output of 70 W for 5 sec, with intermittent cooling of 15 sec. Disruption of cell membrane of all monocytes usually required a total of 15 sec of

KEISUKEWADA e t al.

694

Table I. The dissociation constant (Kd) and maximal binding sites (Bin)of estrogen type I and type II binding sites (mean __+SEM, n = 5) in monocytes in the follicular phase Kd (xI0 ~M) (n=5) B~, (fmol//~g DNA) (n =5) Mean ± SEM.

Type I Type I1 Type I Type II

Cytosol 1.7+0.1 14.3 + 1.8 2.2_+0.8 15.2±2.5

sonication, as seen by a light microscopy after peroxidase staining. The 500/~1 aliquot of this suspension before the centrifugation was used to estimate DNA by the diphenylamine method of Burton (Burton, 1956). This suspension was centrifuged at 800g for 10 min in 4°C to obtain cytosol and crude nuclei. The supernatant was centrifuged at 160,500g for 60 min and this supernatant was exposed in the same volume of 0.3% dextran-coated charcoal (0.003% dextran and 0.3% Noritt A: DCC) solution suspended in TEG buffer to 2-4 x 106cells/ml. The pellet was suspended in TEG buffer and centrifuged at 800g for 10min. The supernatant was decanted and this procedure was done twice. The nuclear KCI extracted fraction was prepared according to the method of Horwitz and Mcguire (1978). The pellet was suspended in 0.5 ml of KC1 buffer (10raM Tris-HCl, 1.5mM EDTA, 10raM thioglycerol, 10% glycerol, 0.6 M KC1, pH 8.5) and incubated at 4°C for I hr during which time the pellet was suspended every 15 min. Then the solubilized proteins were obtained by centrifugation at 90,300g for 30 min. This supernatant was exposed in the same volume of 0.3% DCC solution at 4°C for 1 hr. After centrifugation at 800g for 10min, the supernatant was diluted with 5ml TEG buffer to 1.5-3x 106cells/ml as nuclear KCI soluble fraction. The 90,300g pellet was washed twice with TEG buffer and suspended in l ml TEG buffer and disrupted by sonication in a Heat System Sonicator at an output of 70 W for 5 sec, with intermittent cooling of 15 sec. Nuclear KCI insoluble fraction was collected to filtrate by nylon mesh (100/zm) and the filtrate was suspended with 5ml TEG buffer.

Saturation analysis of [JH]Ez binding Saturation analysis of estrogen binding in each fraction was performed in the presence of [3H]E2 of final concentrations ranging from 1.0-40nM. Cytosol, nuclear KCI soluble fraction and nuclear KCI insoluble suspension (I00#1) were added to two series of tubes. One series contained [3H]~ and 200-fold molar excess of DHT, while the second series was identical except that it contained a 200-fold molar excess of DES. DHT was added to remove the effects of steroid hormone binding globulin. Tubes were incubated at 4°C for 16 hr and specific binding was determined by dextran-coated-charcoal assay for cytosol and nuclear KCI soluble fraction and by sedimentation assay for nuclear KCI insoluble fraction. Specific binding quantity was the difference of bindings in the absence and presence of 200-fold excess of DES for [3H]E2. Data was plotted according to Scatchard (1) and the dissociation constant (Kd) and the maximum binding sites (Bin) of type I binding sites were calculated. The Kd for the type II binding sites was

KCI extract 1.8_+0.2 16.5 ± 2.9 3.7±0.5 14.4_+ 1.3

KCI non-extract 3.0+0.6 17.7±3.9 17.2+5.2 52.0_+8.0

determined as the concentration of [3H]E, required to half saturable type II sites. Bm of these sites was estimated as the [JH]E z specific binding at the total concentration of 40 nM [3H]E2, because these KdS were usually under 20 nM.

In vitro competition assay [3H]Ez (I or 40 nM) with 200-fold excess of DHT and increasing concentration of danazol (0-10~-fold excess) with each 0. I ml ofcytosol, nuclear KCI soluble fraction and KCI insoluble fraction was incubated for 16 hr at 4°C in triplicate. Free forms were removed by DCC in soluble fractions and by pelleting in a insoluble fraction.

Counting of radioactivity The radioactivity of the bound form was determined in aqueous counting scintillant II (Amersham Corporation, Arlington Heights, Ill.) with a Packard Tri-Carb 460C automatic scintillation system (Packard Instrument Company, Downers Grove, Ill.).

Statistics All data are presented as the mean + SEM. Statistical analysis was performed with the Newman-Keuls test, regression analysis and Student's t-test. RESULTS

Estrogen binding characteristics in monocytes from normal women during follicular phase and effect of danazol on estrogen binding T h e binding characteristics o f [3H]E2 in cytosoi, nuclear KCI soluble a n d nuclear KCI insoluble fractions o f m o n o c y t e s p r e p a r e d immediately after harvest from n o r m a l w o m e n are s h o w n in Fig. I. T h e s a t u r a t i o n curves were sigmoidal. S c a t c h a r d a n d Hill analyses d e m o n s t r a t e d the presence o f estrogen type I a n d type II binding sites were d e m o n s t r a t e d . T h e s u m m a r y o f dissociation c o n s t a n t (Kd) a n d m a x i m a l binding sites (Bin) o f estrogen type I a n d type II binding sites are s h o w n in T a b l e 1. D a n a z o l in various c o n c e n t r a t i o n s tested inhibited i n M [~H]E z binding slightly, but 2 0 n M [3H]Ez binding moderately which was the representative determined in the nuclear KCI insoluble fraction o f m o n o c y t e s (Fig. 2). These d a t a indicate t h a t estrogen type I a n d type II binding sites are present in the peripheral monocytes, a n d t h a t d a n a z o l in high c o n c e n t r a t i o n c a n inhibit estrogen binding to those sites.

Fig. 1 (facing page). Saturation, Scatchard and Hill analyses of estrogen binding in monocytes of normal women's peripheral blood prepared immediately after harvest. Total binding is indicated as [3H]F,2 plus a 200-fold excess of DHT (O 0 ) . Non-specific binding is indicated as [3H]Ez plus a 200-fold excess of both DHT and DES ( A A). The presence of two estrogen binding sites in monocytes was seen. (a) Cytosol fraction, (b) nuclear KCI soluble fraction, (c) nuclear KCI insoluble fraction.

EBS in monocytes and effects of danazol in vitro

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Fig. 2. Effects of increasing concentration from l- to 1000-fold excess of danazol and DES on [3H]E2 binding in nuclear KCI insoluble fraction of monocyte. (a) Estrogen type I binding in 0. I nM [3H]E2.(b) Estrogen type !I binding in 40 nM [3H]E:. Each value is the mean of triple determinations in triplicate. This experiment was done in the nuclear KCI insoluble fraction because of the abundance of estrogen binding sites. Effects o f danazol on estrogen binding sites in monocytes in vitro

There were two estrogen binding sites in their peripheral monocytes incubated with MEM medium (Fig. 3) and/or danazol (Fig. 4) for 24 hr at 37°C and 5% CO: atmosphere. The Bm of estrogen binding sites of monocytes incubated with only MEM medium for 24 hr was significantly higher than that of monocytes harvested immediately, while the Kd was similar (data not shown). The reason for this phenomenon was unknown, it may correlate with the differentiation of macrophage. Effects of danazoi on estrogen type I binding sites in cytosol, nuclear KCI soluble fraction and KCI insoluble fraction are shown in Fig. 5 and those on estrogen type II binding sites are in Fig. 6. The Bins of type I sites were significantly decreased by danazol KCI soluble fraction (P < 0.05). Maximal binding sites of type II were decreased by danazol in KCI insoluble fraction significantly (P < 0.05). These results indicate that in high concentration danazol can bind to estrogen binding sites, especially type II sites and decreases both estrogen binding sites in peripheral monocytes /n vitro. DISCUSSION

The monokines exist in peritoneal fluid of patients with pelvic endometriosis, and contribute to

infertility (Hill et al., 1987). Macrophages are derived from peripheral blood monocytes, which are the targets of estrogen (Polan et al., 1988) and have estrogen binding sites (Ranelleti et al., 1988). Both E 2 and progesterone stimulate II-li/ and TNF productions in OK-432-stimulated human peripheral blood monocytes in physiological concentration and inhibit them in the pharmacological concentration. Danazol inhibits those productions in any concentration and dose-dependently (Mori et al., 1992). Danazol binds to androgen receptor, but not much to estrogen receptor in target cells. However danazol, in high concentration, can bind to estrogen binding sites and cause anti-estrogen action (Tamaya et al., 1983). It is possible that danazol can demonstrate the effect on the monokine secretion through the interference to estrogen receptor mechanism. Therefore, the present study showed estrogen binding sites in the peripheral blood monocytes and effects of danazol on those in vitro. The presence of estrogen type I and type II binding sites in peripheral blood monocytes was demonstrated in the human subjects. Two types of estrogen binding sites have previously demonstrated in rat uterus (Clark et al., 1979), rabbit uterus and human endometrium (Wada, 1986). Estrogen type I binding sites are identical with classical estrogen receptor. Estrogen affects the function of human blood mononuclear cells to modulate immune responses through type I binding sites (Weusten et al., 1986). The biological role of estrogen type II binding sites has been proposed to be implicated in the long-term growth effects of E2 in the uterus (Clark et al., 1979). Whelly et al. demonstrated these sites in the rat uterine nucleoli and indicated the close correlation between the stimulation of nucleolar RNA synthesis and the increase of type II sites at the late phase events after administration of E2 at this phase. Namely, nuclear type II sites involved in the late uterine response of estrogen, i.e. uterine growth (Whelly, 1986). While human blood mononuclear cells do not need to grow and proliferate, the role of the estrogen type II binding sites has been speculated to be the metabolic machinery of mononuclear cells, such as taking up retaining large amounts of estradiol in the cells (Ranelleti et al., 1988). This study shows that the presence of estrogen type I and type II binding sites in peripheral blood monocytes in follicular phase and danazol decreases these sites m vitro. The presence of estrogen binding sites of macrophages is relevant to the evidence that the release of IL-I from macrophage was stimulated by estrogen in the physiological concentration in vitro. Progesterone in the physiological concentration increases IL-I production in the monocytes. However, progesterone receptor in the monocytes, was not detected (data not shown). In the high progesterone concentration (10-7M), IL-I production of the

Fig. 3 (facing page). Saturation analyses of estrogen binding in monocytes of normal women's peripheral blood after incubation with MEM medium for 24 hr. Total binding is indicated as [3H]Ezplus a 200-fold excess of D H T (O '" 0). Non-specific binding is indicated as [3H]E2plus a 200-fold excess of DHT and DES (& &). (a) Cytosol fraction, (b) nuclear KCI soluble fraction, (c) nuclear KCI insoluble fraction.

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